Cardiovascular disease is a major cause of mortality and morbidity and development of therapies that address its underlying causes is an important public health priority. With better management of LDL-C through statin therapy, low HDL-C and its attendant dysregulated metabolism have emerged as medical challenges for which current therapies are inadequate. HDL-phospholipids (PL) are essential to reverse cholesterol transport (RCT), the putative cardioprotective mechanism by which peripheral tissue cholesterol is transported into the plasma for hepatic disposal. There is compelling evidence, including studies with reconstituted (r) HDL, that raising HDL-PL enhances multiple steps in RCT and that increased HDL phospholipidation is therapeutic. We discovered two mechanisms that increase HDL-phospholipid. 1] Serum opacity factor (SOF), a protein secreted by S. pyogenes, destabilizes and selectively delipidates human HDL through a disproportionation mechanism that yields a cholesteryl ester-rich microemulsion (CERM), lipid-free-apo-A-I and a phospholipid-rich "neo HDL." 2] HDL can be superphospholipidated (SPLd) by a detergent removal method that we developed. Both processes greatly increase HDL-phosphatidylcholine in a way that increases HDL cholesterophilicity, LCAT reactivity, and cellular cholesterol efflux. To move these discoveries to human therapy requires additional tests in cellular models of cholesterol transport and in mouse models of lipid metabolism and atherogenesis. Our broad goal is to translate these discoveries into new therapeutic modalities for cardioprotection via improved RCT. This will be achieved by completion of four aims. 1: Measure cholesterol efflux from macrophages and cell models of cholesterol efflux to determine the importance of SR-BI, ABCA1, and ABCG1 in efflux to rHDL or HDL with various levels of phospholipidation achieved via SOF or SPLn. 2: Compare the rates of CE uptake of HDL formed by the sequential actions of SOF or SPLn and LCAT on HDL or rHDL in hepatocytes from WT and SR-BI KO mice;measure CERM-CE uptake by hepatocytes from WT and LDL-receptor KO mice. 3: Compare the transfer of peritoneal macrophage-cholesterol to plasma, liver and feces in mice treated with SPLd-HDL or neo HDL with those treated with native HDL, rHDL, and control saline;measure transfer of CERM-[3H]CE to liver, bile, and feces. 4: Compare lesion formation in apo E KO mice infused with native HDL, rHDL, or saline with those infused with SPLd HDL or neoHDL. Enhancing RCT is the next frontier in lipoprotein therapeutics. Completion of our aims will pave the way to translating this basic research into clinical trials on safety and lesion regression using intravascular ultrasound and/or MRI. PUBLIC HEALTH RELEVANCE: Low plasma high density lipoproteins (HDL)-cholesterol, due to impaired reverse cholesterol transport , is a serious lipid disorder for which current treatments are inadequate. We discovered two processes - HDL opacification and HDL superphospholipidation - that enhance multiple steps in RCT in vitro and plan to move these toward clinical management of dysregulated HDL metabolism by determining whether they improve RCT and inhibit and/or reverse atherosclerosis in cell and mouse models of lipid disorders and atherosclerosis. Completion of our aims would address an important public health priority by validating two new therapeutic modalities.